Resolution of racemates of primary and secondary heteroatom-substitued amines by enzyme-catalyzed acylation

ABSTRACT

A process for preparing optically active primary and secondary heteroatom-substituted amines from the corresponding racemates, which comprises 
     a) enantioselective acylation of a racemic heteroatom-substituted amine with an ester whose acid component carries a fluorine, nitrogen, phosphorus, oxygen or sulfur atom in the vicinity of the carbonyl carbon, in the presence of a hydrolase, 
     b) separation of the mixture of optically active heteroatom-substituted amine and optically active acylated heteroatom-substituted amine to obtain one enantiomer of the heteroatom-substituted amine, 
     c) if required isolation of the other enantiomer of the heteroatom-substituted amine from the acylated heteroatom-substituted amine by amide cleavage.

The present invention relates to a novel process for resolving racematesof primary and secondary heteroatom-substituted amines by reaction withan ester in the presence of a hydrolase and subsequent separation of theenantioselectively acylated heteroatom-substituted amine from the other,unreacted, enantiomer of the heteroatom-substituted amine.

WO 95/08636 describes a process for resolving racemates of primary andsecondary amines by reaction with an ester in the presence of ahydrolase. The preferred amines mentioned therein are primaryarylalkylamines. However, there is no reference to the usability ofheteroatom-substituted amines.

We have now found, surprisingly, that the process described at theoutset functions particularly advantageously when theheteroatom-substituted amine used is an amine of the general formula I

where

n is 0, 1, 2, 3;

Y is O, S, NH, NR⁵;

R¹, R² are each, independently of one another, H, alkyl, or aryl or R¹and R² or R² and R³, or R¹ and R⁴ are, together with the adjacent carbonatoms, part of a ring system;

R⁴ is alkyl or arylalkyl;

R³, R⁵ are, independently of one another, H, alkyl or arylalkyl.

We have also found a process for preparing acylated primary andsecondary amines by reacting the heteroatom-substituted amines with anester with specific catalysis by a hydrolase, wherein the acid componentof the ester carries a fluorine, nitrogen, phosphorus, oxygen or sulfuratom in the vicinity of the carbonyl carbon.

The esters suitable for the process according to the invention are thosewhich carry in the acid component of the ester an electron-richheteroatom in the vicinity of the carbonyl carbon or in which anacceptor substituent in the form of one or more heteroatonms is locatedin the vicinity of the carbonyl carbon in the acid component.

The heteroatom must have at least one free pair of electrons. It can bea fluorine, nitrogen, phosphorus, oxygen or sulfur atom.

It should be located in the vicinity of the carbonyl carbon. This meansthat the heteroatom is bonded to a carbon atom in the position alpha,beta or gamma to the carbonyl carbon. The heteroatom can also bemultiply bonded to the carbon as in the cyano group. Preferred acidcomponents in the ester are those in which the heteroatom is bonded tothe alpha carbon atom. Oxygen is preferred as heteroatom.

The heteroatom may also be linked to other groups, eg. alkyl groups. Ifthe heteroatom is, for example, oxygen, an ether moiety is present.

Particularly suitable esters are those having the structure

where

R¹=C₁-C₁₀-alkyl,

R²=C₁-C₁₀-alkyl, H

R³=H, C₁-C₁₀-alkyl, or phenyl which is unsubstituted or substituted byNH₂, OH, C₁₋₄-alkoxy or halogen,

X=O, S, NR⁴,

R⁴=H, C₁-C₁₀-alkyl, or phenyl which is unsubstituted or substituted byNH₂, OH, C₁₋₄-alkoxy or halogen,

n=0, 1 or 2.

Of these, the C₁₋₄-alkyl esters of C₁₋₄-alkoxyacetic acids arepreferred, such as ethyl methoxyacetate.

A large number of enzymes can be used as hydrolases in the processaccording to the invention. Proteases and, in particular, lipases, arepreferably used. Particularly suitable lipases are microbial lipaseswhich can be isolated, for example, from yeasts or bacteria.Particularly suitable lipases are those from Pseudomonas, eg. Amano P orthe lipase from Pseudomonas spec. DSM 8246.

Further particularly suitable hydrolases are the enzymes commerciallyobtainable from Novo Nordisk (Enzyme Toolbox), in particular lipases SP523, SP 524, SP 525, SP 526 and Novozym® 435. These enzymes aremicrobial lipases which can be prepared from yeasts such as Candidaantarctica.

It is furthermore possible and advantageous to employ the lipases“Chirazyme L1 bis L8′, which are commercially obtainable (BoehringerMannheim) in the process according to the invention.

The enzyme can be employed in native or immobilized form. Theimmobilized enzyme Novozym® 435 is particularly suitable.

The processes according to the invention can be carried out in thepresence or absence of solvents.

Organic solvents are generally suitable as solvents. The reaction takesplace particularly well in ethers, for example in MTBE, 1,4-dioxane orTHF, or in hydrocarbons such as hexane, cyclohexane, toluene orhalogenated hydrocarbons such as methylene chloride.

The reaction of the ester with the racemic heteroatom-substituted aminewith enzyme catalysis is normally carried out at room temperature. Thetimes for this reaction are from 1 to 48 hours, depending on thesubstrate. Secondary heteroatom-substituted amines usually requirelonger reaction times than do primary heteroatom-substituted amines. Thelower reactivity of secondary heteroatom-substituted amines can also becompensated by increasing the amount of catalyst by comparison withprimary heteroatom-substituted amines.

0.5-3 mol of ester are added per mol of amine to be reacted. 0.5-3,preferably 0.5-1.0, mol of ester are added even when racemic substratesare used.

The amount of enzyme to be added depends on the nature of the hydrolaseand the activity of the enzyme preparation. The optimal amount of enzymefor the reaction can easily be determined by simple preliminary tests.As a rule, 1000 units of lipase are added per mmol ofheteroatom-substituted amine.

The progress of the reaction can easily be followed by conventionalmethods, for example by gas chromatography. In the case of racemateresolution, it is sensible to terminate the reaction when 50% of theracemic heteroatom-substituted amine is reacted.

This normally takes place by removing the catalyst from the reaction,for example by filtering off the enzyme.

The enantioselective reaction of the racemic substrate with the esterresults in the correspondingly acylated product (amide) from oneenantiomer, while the other enantiomer remains unchanged. The resultingmixture of heteroatom-substituted amines and amide can easily beseparated by conventional methods. For example, extraction ordistillation processes are very suitable for separating the mixture ofamine and amide.

The process according to the invention is particularly advantageouslysuitable for acylating heteroatom-substituted amines of the formula I.It can also be used to resolve racemates of virtually all primary andsecondary heteroatom-substituted amines. It takes place particularlywell with primary amino alcohols, especially those in which R⁴ isarylalkyl, in particular benzyl, or alkyl, in particular methyl.

Further preferred compounds of the formula I are those where R¹ and R²form with the adjacent carbon atoms a ring system, in particular thoseof the following structure

or R² and R³ are part of a ring system, in particular those of thefollowing structure

or R¹ and R⁴ are part of a ring system, in particular those of thefollowing structure

Surprisingly, the reaction of heteroatom-substituted amines of theformula I takes place with very much higher optical yields than thesimilar reaction of non-heteroatom-substituted amines or thosesubstituted differently from formula I.

Furthermore, as a consequence of the high selectivity and reactivity ofthe process according to the invention, only a small, or no, excess ofacylating agent is needed, which greatly facilitates subsequentseparation and purification.

The invention is also suitable for preparing optically active primaryand secondary heteroatom-substituted amines from the correspondingracemates, by

a) enantioselective acylation of a racemic heteroatom-substituted aminewith an ester whose acid component carries a fluorine, nitrogen, oxygenor sulfur atom in the vicinity of the carbonyl carbon, in the presenceof a hydrolase,

b) separation of the mixture of optically active heteroatom-substitutedamine and optically active acylated heteroatom-substituted amine toobtain one enantiomer of the heteroatom-substituted amine,

c) if required isolation of the other enantiomer of theheteroatom-substituted amine from the acylated heteroatom-substitutedamine by amide cleavage.

The process according to the invention can be made even more economicif, after removal of the required enantiomer, the remaining unwantedenantiomer is racemized and employed anew in the process. This recyclingmakes it possible to obtain a total of more than 50% of the requiredenantiomer from the racemic heteroatom-substituted amine.

Not only are the processes according to the invention suitable forproducing optically active primary and secondary heteroatom-substitutedamines, they can also form part of complicated multi-stage chemicalsyntheses, for example in the preparation of medicinal agents or cropprotection agents.

The following examples illustrate the invention.

EXAMPLE 1 General Method for the Lipase-catalyzed Acylation ofHeteroatom-substituted Amines

10 mmol of the primary or secondary heteroatom-substituted amine aredissolved in MTBE (=methyl tert-butyl ether) (about 10% strengthsolution). 11 mmol of ethyl methoxyacetate are added to the solution andthe reaction is started by adding 100 mg of lipase (about 1000 U/mg,Pseudomonas spec. DSM 8246). When the reaction is complete (12-48 h,depending on the heteroatom-substituted amines), the enzyme is filteredoff, and the solution is concentrated under reduced pressure. Themethoxyacetamides are obtained in a yield of more than 90%.

EXAMPLE 2 General Method for Racemate Resolution

The primary or secondary heteroatom-substituted amine is dissolved inMTBE (about 10% by weight). Addition of 1 mol of ethyl methoxyacetateper mol of racemic heteroatom-substituted amine is followed by that ofPseudomonas lipase (DSM 8246) and the suspension is stirred at roomtemperature. About 10,000 units of lipase (10 mg) are added per mmol ofheteroatom-substituted amine. After 50% reaction has occurred (checkedby gas chromatography), which takes 1-48 h depending on theheteroatom-substituted amines, the enzyme is filtered off. The mixtureof heteroatom-substituted amines and acylated heteroatom-substitutedamine (amide) is separated by distillation or extraction.

EXAMPLE 3 Racemate Resolution with Solvent

5 g (49.5 mmol) of trans-2-aminocyclopentanol were dissolved in 20 ml of1,4-dioxane, 3.3 g (25 mmol) of isopropyl methoxyacetate were added and,after addition of 0.1 g of Novozym 435®, shaken at room temperature.After 12 h, ¹H-NMR showed 50% reaction of amine; the enzyme was filteredoff, the filtrate was concentrated, and the unreacted amine was removedfrom the amide by distillation.

Yields

EXAMPLE 4 Racemate Resolution Without Solvent

5 g (2 mmol) of trans-2-benzyloxy-1-cyclopentylamine and 1.8 g (13.4mmol) of isopropyl methoxyacetate were mixed, 0.1 g of Novozym 435® wasadded, and the mixture was shaken at room temperature. ¹H-NMR showed 50%reaction of amine after 120 h. The enzyme was filtered off and the‘amine’ was separated from the ‘amide’ by extraction with 10% strengthhydrochloric acid.

Yields

EXAMPLE 5 Further Racemate Resolutions

The following reactions (see Table) were carried out as in Example 3 or4.

TABLE Starting Preparation Conversion Amine Amide material as in Example[%] Rotation* ee Rotation* ee

3 50 −1.5° (c = 1 in ethanol) 24% — 26% (by HPLC)

3   4 50   50 +4.9°(HCl adduct) (c = 1 in CHCl₃) +4.8°(HCl adduct) (c =1 in CHCl_(3 +9.8° (c = 1 in dioxane)) >95% (by HPLC)     >95% (by HPLC)+28° (c = 1 in dioxane)     +27.5° c = 1 in dioxane >99.5% (by HPLC)    >99.5% (by HPLC)

4 42 70% — 99.5% (by HPLC) *The rotations were measured with the Na-Dline at room temperature.

The table in Example 5 shows that very much higher optical purities canbe obtained on use of ‘protected’ amino alcohols in which the oxygenatom is, for example, adjacent to a benzyl or methyl group than on useof unprotected amino alcohols.

We claim:
 1. A process for preparing acylated primary and secondaryoxygen or nitrogen substituted amines by reacting the oxygen or nitrogensubstituted amines with an ester of the formula:

where R₁=C₁-C₁₀-alkyl, R²=C₁-C₁₀-alkyl or H, R³=H, C₁-C₁₀-alkyl, orphenyl which is unsubstituted or substituted by NH₂, OH, C¹⁻⁴-alkoxy orhalogen, in the presence of a lipase selected from the group consistingof SP 523, SP 524, SP 525, SP 526 and Novozym®
 435. 2. The process ofclaim 1 wherein the oxygen or nitrogen substituded amine reacted is acompound of the formula I:

where n is 0, 1, 2, 3; Y is O, NH, or NR⁵; R¹, R² are each,independently of one another, H, alkyl, or aryl or R¹ and R², or R² andR³, or R¹ and R⁴ are, together with adjacent carbon atoms, part of aring system; R⁴ is alkyl or arylalkyl; R³, R⁵ are, independently of oneanother, H, alkyl or arylalkyl.
 3. The process of claim 2 wherein, inthe ester, R¹ is C₁-C₄-alkyl and R² is C₁-C₄-alkyl.
 4. The process ofclaim 3 wherein in the ester is ethyl methoxyacetate.
 5. A process forresolving racemates of primary and secondary oxygen or nitrogensubstituted amines by reacting the oxygen or nitrogen substituted amineswith an ester of the formula:

where R¹=C₁-C₁₀-alkyl, R²=C₁-C₁₀-alkyl or H, R³=H, C₁-C₁₀-alkyl, orphenyl which is unsubstituted or substituted by NH₂, OH, C₁₋₄-alkoxy orhalogen, in the presence of a lipase selected from the group consistingof SP523, SP524, SP525, SP526 and Novozym® 435 and subsequentlyseparating the oxygen or nitrogen substituted amine, which has undergoneenantioselective acylation, from the other unreacted enantiomer of theheteroatom-substituted amine.
 6. The process of claim 5 wherein theoxygen or nitrogen substituted amine reacted is a compound of theformula I:

where n is 0, 1, 2, 3; Y is O, NH, or NR⁵; R¹, R² are each,independently of one another, H, alkyl, or aryl or R¹ and R², or R² andR³, or R¹ and R⁴ are, together with adjacent carbon atoms, part of aring system; R⁴ is alkyl or arylalkyl; R³, R⁵ are, independently of oneanother, H, alkyl or arylalkyl.
 7. The process of claim 6 wherein, inthe ester, R¹ is C₁-C₄-alkyl and R² is C_(1-C) ₄-alkyl.
 8. The processof claim 7 wherein in the ester is ethyl methoxyacetate.
 9. A processfor preparing optically active primary and secondary oxygen or nitrogensubstituted amines by a) reacting the oxygen or nitrogen substitutedamines with an ester of the formula:

 where R¹=C₁-C₁₀-alkyl, R²=C₁-C₁₀-alkyl or H, R³=H, C₁-C₁₀-alkyl, orphenyl which is unsubstituted or substituted by NH₂, OH, C¹⁻⁴-alkoxy orhalogen, in the presence of a lipase selected from the grout consistingof SP523, SP524, SP525, SP526 and Novozym®435, and b) separating of themixture of optically active oxygen or nitrogen substituted amine andoptically active acylated oxygen or nitrogen substituted amine to obtainone enantiomer of the oxygen or nitrogen substituted amine.
 10. Theprocess of claim 9 wherein the oxygen or nitrogen substituted aminereacted is a compound of the formula I:

where n is 0, 1, 2, 3; Y is O, NH, or NR⁵; R¹, R² are each,independently of one another, H, alkyl, or aryl or R¹ and R², or R² andR³, or R¹ and R⁴ are, together with adjacent carbon atoms, part of aring system; R⁴ is alkyl or arylalkyl; R³, R⁵ are, independently of oneanother, H, alkyl or arylalkyl.
 11. The process as claimed in claim 10,wherein step b) is followed by another step in which the unwantedenantiomer is racemized and subsequently returned to the racemateresolution process.
 12. The process of claim 10 wherein the otherenantiomer of the amine from the acylated oxygen or nitrogen substituteis separated by amide cleavage.
 13. The process of claim 12 wherein, inthe ester, R¹ is C₁-C₄-alkyl and R² is C₁-C₄-alkyl.
 14. The process ofclaim 13 wherein in the ester is ethyl methoxyacetate.
 15. The processof claim 12 wherein the amide cleavage is followed by another step inwhich the unwanted enantiomer is racemized and subsequently returned tothe racemate resolution process.